Method and device for detecting a biofilm

ABSTRACT

A method for determining the thickness of a biofilm which is developed on a support which is immersed in an aqueous medium in which a biofilm develops, characterized in that it consists in the following: a) the medium is continuously circulated in an electrochemical cell comprising a reference electrode, an auxiliary electrode and at least one working electrode, b) circulation of said medium is interrupted and the cell is isolated; c) an electrochemical tracer is introduced into the medium present in the cell; d) the medium/tracer solution is circulated in the cell in order to direct a jet of said solution, perpendicular to the working electrode; e) the value of the tracer reduction limiting current is measured and recorded according to the hydrodynamic conditions on the surface of the working electrode; and f) the thickness of the porous layer of biofilm on the surface of the electrode is calculated according to the Koutecky-Levich relation, based on analysis of the transport of material through the porous layer which links the value of the tracer reduction limiting current to the solution flow.

FIELD OF THE INVENTION

The present invention relates to a method and a device for determiningthe thickness of a biofilm.

BACKGROUND OF THE INVENTION

It is known that, especially in the field of water treatment, fixedculture techniques are employed in which the ability of microorganismsto produce exopolymers enabling them to be fixed to very diversesupports in order to form biofilters is used. Although, in theseapplications, the properties of the biofilms are exploited beneficiallyto remove contaminants (nitrates, phosphates, etc.), on the other hand,the same biofilms are often undesirable, or even detrimental. Thus, insome industrial plants immersed in aqueous media, the development ofmicroorganisms, bacteria and algae, which are deposited on the plants inthe form of biofilms, constitutes real microbiological pollution and theprevention and control of the growth of these biofilms requireimplementing relatively sophisticated, efficient and expensivetechniques, the most common of which is chlorination.

Optimization of the treatments (improving performance, removing waste,reducing costs) is related to the possibility of determining the growthrate of the biofilms in real time.

At present, methods making it possible to detect the presence ofbiofilms and which have been the subject of industrial development arefew. In order to illustrate the prior art in this field mention mayespecially be made of the following publications.

U.S. Pat. Nos. 4,912,332 and 5,337,376 relate to a method of detectingmicrobiological pollution using optical fibres. This known technique isbased on measuring transmitted and absorbed light in order to monitorthe formation of the biofilms. It has the drawback of being difficult touse in pipes.

U.S. Pat. No. 5,576,481 describes a method of detecting on-line biofilmsbased on measuring the heat transfer coefficient. The change in thelatter is directly related to the development of the biofilm.

An apparatus making it possible to identify the biofilms and to controlthe use of biodispersants, which uses the conventional method ofmeasuring the adenosine triphosphate is known by the brand “Bioscan”.This apparatus has in particular the following drawbacks: on the onehand, the measurement depends on the bacterial cells sampled in themedium studied and not on the biofilm itself and, on the other hand, itis complicated and expensive.

Another sensor for monitoring the biofilm, called “BIoGEORGE” is known,which consists of two stainless steel electrodes, mounted on a body alsomade of stainless steel, and a data control and acquisition system. Themeasurement is based on analysing the change in current flowing betweenthe two electrodes, after interruption by a galvanostaticprepolarization. This change is correlated to the presence of thebiofilm and to the type of corrosion products on the surface of theelectrodes. Given that the interaction between the biofilm and thestainless steels is very complex, detection of the biofilm alone seemsvery difficult with this sensor and, in addition, the measurements canonly be carried out in seawater.

The “BioX” sensor records the value of the galvanic coupling currentbetween a stainless steel and a copper alloy which is associated withthe growth phase of the biofilm.

The “BioGuard” sensor, based on the electrochemical detection of thecatalysis for the reduction of oxygen by bacteria, makes it possible tomonitor the first steps in the formation of the biofilm.

B. N. Stokes et al (“Developments in on-line fouling and corrosionsurveillance” published in “Microbiologically influenced corrosiontesting” in 1994 by J. R. Keans and B. Little, Philadelphia, USA) havedeveloped a device enabling corrosion and fouling to be monitored havingthe form of a miniaturized heat exchanger in which the corrosion ismonitored by a measurement using an ammeter with zero resistance, ameasurement of the electrochemical noise (current/potential) and ameasurement of the linear polarization resistance, while fouling isdetected by measuring the heat transfer coefficient. The use of thesefour electrochemical techniques makes the system complex and not veryadaptable on site.

G. Salvago et al (“Biofilm monitoring and on-line control: 20-monthexperience in seawater” published in 1994 in “Microbiol Corrosion” bythe European Federation of Corrosion) have studied the behaviour ofstainless steels and of aluminium brasses exposed to seawater. Thecombination of the heat transfer resistance and of the electrochemicalmeasurements under cathodic polarization enables the growth of a biofilmon the inner surface of tubes to be monitored. The various techniquesused in such a system make it complex.

Experience demonstrates that all the currently known biofilm detectorshave the drawback of being complex in their use. The present inventiontherefore set itself the objective of providing a biofilm sensor makingit possible to measure the thickness of the biofilm and which can beused in various media (seawater, freshwater, water from industrialprocesses, etc.).

According to the present invention, the ability of a given medium todevelop a biofilm when travelling around a circuit is assessed byelectrochemical means by using a sensor based on the principle of theelectrochemical cell with three electrodes.

Firstly, and according to a first aspect, this invention aims to providea method of determining the thickness of a biofilm developing on asupport immersed in an aqueous medium where such a biofilm isdeveloping, characterized in that it consists in:

-   -   a) continuously circulating the said medium in an        electrochemical cell comprising a reference electrode, an        auxiliary electrode and at least one working electrode;    -   b) interrupting the circulation of the said medium and isolating        the cell;    -   c) introducing an electrochemical tracer in the medium present        in the cell;    -   d) circulating the medium+tracer solution in the said cell so as        to direct a jet of the said solution perpendicularly to the        working electrode;    -   e) measuring and recording the value of the limiting current for        reducing the said tracer, as a function of the hydrodynamic        conditions at the surface of the working electrode, and    -   f) calculating the thickness of the porous biofilm layer at the        surface of the said electrode by applying the Koutecky-Levich        equation, based on analysing the transport of matter through the        porous layer which relates the value of the limiting current for        reducing the tracer to the flowrate of the solution.

With regard to the Koutecky-Levich equation, reference may be made tothe article by D. Herbert-Guillou et al published in ElectrochimicaActa, 1999, Vol. 4, No. 7, page 1067.

BRIEF DESCRIPTION OF THE INVENTION

According to a second aspect, the invention also aims to provide amethod of determining the thickness of a biofilm developing on a supportimmersed in an aqueous medium where such a biofilm is developing,characterized in that it consists in:

-   -   a) continuously circulating the said medium in an        electrochemical cell comprising a reference electrode, an        auxiliary electrode and at least one working electrode;    -   b) interrupting the circulation of the said medium and isolating        the cell;    -   c) circulating, in the said cell, the said medium in which        oxygen is normally dissolved and which functions as an        electrochemical tracer, so as to direct a jet of the said medium        perpendicularly to the working electrode;    -   d) measuring and recording the value of the limiting current for        reducing the oxygen dissolved in the medium, as a function of        the hydrodynamic conditions at the surface of the working        electrode, and    -   e) calculating the thickness of the porous biofilm layer at the        surface of the said electrode by applying the Koutecky-Levich        equation, based on analysing the transport of matter through the        porous layer which relates the value of the limiting current for        reducing the tracer to the flowrate of the solution.

Thus according to the inventive method, the value of the limitingcurrent for reducing an electrochemical tracer, which can for example bethe oxygen present in the medium or potassium ferricyanide introducedinto the medium contained in the cell before the measurement, as afunction of the hydrodynamic conditions at the surface of the workingelectrode, is recorded. The Koutecky-Levich equation based on analysingthe transport of matter through a porous layer relates the value of thelimiting current for reducing the tracer to the flowrate of theelectrolytic solution (medium+tracer) and makes it possible to calculatethe thickness of the porous layer at the surface of the workingelectrode.

Secondly, the invention aims to provide a device for implementing themethod defined above which is characterized in that it comprises:

-   -   an electrochemical cell fitted with a reference electrode, a        counter electrode and at least one working electrode, the said        cell defining a chamber receiving the pipes for supply and        discharge of the medium to be studied, with the insertion of        solenoid valves;    -   a recycling loop in which the electrolytic solution consisting        of the medium to be studied, to which the electrochemical tracer        has been added, or in which the naturally dissolved oxygen acts        as an electrochemical tracer, circulates continuously during the        measurement;    -   a set of nozzles, each one placed opposite a working electrode        in order to direct a jet of the electrolytic solution        perpendicularly onto the said electrodes;    -   a variable capacity pump on the said recycling loop, and    -   instrumentation for measuring the thickness of the biofilm which        is deposited on the working electrodes during the circulation of        the medium to be studied through the cell.

Other characteristics and advantages of the invention will becomeapparent from the description given below of an exemplary embodiment ofthe invention given by way of non-limiting example with reference to thedrawings in which:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the device according to theinvention,

FIGS. 2 and 3 are schematic views in plan and in section along 3-3 ofFIG. 2 of a sensor according to the invention, and

FIG. 4 illustrates the mean thickness of a biofilm obtained in naturalseawater, for a flowrate of 6 ml.s⁻¹, as a function of immersion time.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIGS. 1 to 3, it can be seen that, in this exemplaryembodiment, the device according to the invention comprises anelectrochemical cell, denoted overall by the reference 10, whichcomprises a reference electrode 11, a counter electrode 12 and workingelectrodes such as 13. In this exemplary embodiment, six workingelectrodes are provided in order to overcome the spatial heterogeneityof the biofilm. The electrodes preferably consist of the followingmaterials:

-   -   reference electrode 11: calomel (Hg/Hg₂Cl₂)    -   counter electrode 12: platinized platinum,    -   working electrodes 13: gold.

The medium to be studied, in particular seawater, freshwater or waterfrom an industrial process, is introduced into the cell via a centralnozzle 14 and it emerges therefrom via pipes such as 15, solenoid valvesbeing provided on these inlets and outlets.

A closed-loop circuit 16 is linked to the cell 10. The electrochemicaltracer is introduced into this circuit and the electrolytic solution(medium+tracer) continuously circulates by virtue of a variable capacitypump 18. As can be seen in FIG. 1, the solenoid valves enable the loop16 to be put out of service when the sensor is not used for measuringthe thickness of the biofilm. The loop circuit 16 opens out into thecell via nozzles such as 17 facing each working electrode 13 so as todirect a jet with a variable flowrate of the electrolytic solutionperpendicularly onto the said electrodes.

The device is completed by instrumentation such as a potentiostat andmultimeter for measuring the thickness of the biofilm layer on theworking electrodes such as 13.

This device is implemented as follows:

-   -   1) circulating the medium studied in the cell 10,    -   2) measurement at t=0 with no biofilm on the working electrodes,        -   a) stopping the circulation of the medium, isolating the            cell by closing the solenoid valves on the pipes for supply            and discharge of the medium;        -   b) polarizing the selected working electrode (13) at a            predetermined potential;        -   c) bringing the variable capacity pump 18 into service after            having introduced the electrochemical tracer into the loop            16; stabilizing the flowrate; measuring the current;            changing the value of the flowrate of the pump 18, a new            current measurement; repeating this cycle for a            predetermined number of times;        -   d) shutting down the variable capacity pump;        -   e) stopping the polarization of the electrode;        -   f) passing to the following working electrode; repeating the            previous cycle on this electrode.    -   3) bringing the medium back into circulation in the cell by        opening the solenoid valves on the introduction 14 and discharge        15 pipes. Taking the loop 16 out of service.    -   4) at regular intervals:        -   a) resuming the cycle 2;        -   b) calculating the thickness of the biofilm deposited on the            working electrodes 13.    -   5) bringing the medium back into circulation.

The sensor according to the invention has made it possible to monitorthe change in thickness of the biofilm during ageing in natural seawaterfor a period of forty-three days. After eight days of immersion, abiofilm of a few micrometers thick was detected on the electrodes,reaching several tens of micrometers after forty-three days. The curveillustrated in FIG. 4 illustrates the variation of the mean thickness ofthe biofilm thus obtained as a function of the immersion time, for acirculation flowrate of 6 ml.s⁻¹.

It results from reading the above description that the inventionprovides a device which is simple, inexpensive and easy to use, makingit possible to measure the thickness of the biofilm in different media.

Of course it remains that the invention is not limited to the exemplaryembodiments and/or the implementational examples described and/or shownbut that it encompasses all variants.

1. A method of determining the thickness of a biofilm developing on asupport immersed in an aqueous medium where such a bioflim isdeveloping, comprising the steps: a) continuously circulating the mediumin an electrochemical cell comprising a reference electrode, anauxiliary electrode and at least one working electrode; b) interruptingthe circulation of the medium and isolating the cell; c) introducing anelectrochemical tracer in the medium present in the cell; d) circulatingthe medium+tracer solution in the cell so as to direct a let of thesolution perpendicularly to the working electrode; e) measuring andrecording the value of the limiting current for reducing the tracer, asa function of the hydrodynamic conditions at the surface of the workingelectrode; f) calculating the thickness of the porous bioflim layer atthe surface of the electrode by applying the Koutecky-Levich equation,based on analyzing the transport of matter through the porous layerwhich relates the value of the limiting current for reducing the tracerto the flowrate of the solution; and wherein the electrochemical traceris potassium ferricyanide.
 2. A method of determining the thickness of abiofilm developing on a support immersed in an aqueous medium where sucha biofilm is developing, comprising the steps: a) continuouslycirculating the medium in an electrochemical cell comprising a referenceelectrode, an auxiliary electrode and at least one working electrode; b)interrupting the circulation of the medium arid isolating the cell; c)introducing an electrochemical tracer in the medium present in the cell;d) circulating the medium+tracer solution in the cell so as to direct ajet of the solution perpendicularly to the working electrode; e)measuring and recording the value of the limiting current for reducingthe tracer, as a function of the hydrodynamic conditions at the surfaceof the working electrode; f) calculating the thickness of the porousbioflim layer at the surface of the electrode by applying theKoutecky-Levich equation, based on analyzing the transport of matterthrough the porous layer which relates the value of the limiting currentfor reducing the tracer to the flowrate of the solution; anelectrochemical cell fitted with a reference electrode, a counterelectrode and at least one working electrode, the cell defining achamber receiving pipes for supply and discharge of the medium to bestudied, with the insertion of solenoid valves; a recycling loop inwhich the electrolytic solution consisting of the medium to be studied,to which the electrochemical tracer has been added, or of the mediumitself in which the naturally dissolved oxygen acts as a tracer,circulates continuously during the measurement; a set of nozzles, eachone placed opposite a working electrode in order to direct a jet of theelectrolytic solution perpendicularly onto the electrodes; a variablecapacity pump on the recycling loop, and instrumentation for measuringthe thickness of the biofilm which is deposited on the workingelectrodes during the circulation of the medium to be studied throughthe cell.